Heat inactivation of foetal bovine serum performed after EV‐depletion influences the proteome of cell‐derived extracellular vesicles
Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS‐derived EVs by ultracentrifugation...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2024-01-01
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| Series: | Journal of Extracellular Vesicles |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/jev2.12408 |
| _version_ | 1797346227567722496 |
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| author | Ornella Urzì Markus Bergqvist Cecilia Lässer Marta Moschetti Junko Johansson Daniele D´Arrigo Roger Olofsson Bagge Rossella Crescitelli |
| author_facet | Ornella Urzì Markus Bergqvist Cecilia Lässer Marta Moschetti Junko Johansson Daniele D´Arrigo Roger Olofsson Bagge Rossella Crescitelli |
| author_sort | Ornella Urzì |
| collection | DOAJ |
| description | Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS‐derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell‐derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no‐HI FBS), FBS heat inactivated before EV depletion (HI‐before EV‐depl FBS), and FBS heat inactivated after EV depletion (HI‐after EV‐depl FBS). We isolated large (L‐EVs) and small EVs (S‐EVs) from FBS treated in the three different ways, and we found that the S‐EV pellet from HI‐after EV‐depl FBS was larger than the S‐EV pellet from no‐HI FBS and HI‐before EV‐depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI‐after EV‐depl significantly increased the protein amount of S‐EVs but had no significant effect on L‐EVs. Consequently, the protein quantity of S‐EVs isolated from three cell lines cultured in media supplemented with HI‐after EV‐depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS‐derived S‐EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no‐HI FBS and HI‐before EV‐depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell‐derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co‐isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. |
| first_indexed | 2024-03-08T11:29:59Z |
| format | Article |
| id | doaj.art-5a2ce9ea25c84ec39c16835f4b748ca6 |
| institution | Directory Open Access Journal |
| issn | 2001-3078 |
| language | English |
| last_indexed | 2024-03-08T11:29:59Z |
| publishDate | 2024-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Vesicles |
| spelling | doaj.art-5a2ce9ea25c84ec39c16835f4b748ca62024-01-26T03:51:18ZengWileyJournal of Extracellular Vesicles2001-30782024-01-01131n/an/a10.1002/jev2.12408Heat inactivation of foetal bovine serum performed after EV‐depletion influences the proteome of cell‐derived extracellular vesiclesOrnella Urzì0Markus Bergqvist1Cecilia Lässer2Marta Moschetti3Junko Johansson4Daniele D´Arrigo5Roger Olofsson Bagge6Rossella Crescitelli7Sahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational Medicine, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy University of Gothenburg Gothenburg SwedenKrefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy University of Gothenburg Gothenburg SwedenKrefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy University of Gothenburg Gothenburg SwedenSahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational Medicine, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy University of Gothenburg Gothenburg SwedenSahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational Medicine, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy University of Gothenburg Gothenburg SwedenKrefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy University of Gothenburg Gothenburg SwedenSahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational Medicine, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy University of Gothenburg Gothenburg SwedenSahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational Medicine, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy University of Gothenburg Gothenburg SwedenAbstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS‐derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell‐derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no‐HI FBS), FBS heat inactivated before EV depletion (HI‐before EV‐depl FBS), and FBS heat inactivated after EV depletion (HI‐after EV‐depl FBS). We isolated large (L‐EVs) and small EVs (S‐EVs) from FBS treated in the three different ways, and we found that the S‐EV pellet from HI‐after EV‐depl FBS was larger than the S‐EV pellet from no‐HI FBS and HI‐before EV‐depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI‐after EV‐depl significantly increased the protein amount of S‐EVs but had no significant effect on L‐EVs. Consequently, the protein quantity of S‐EVs isolated from three cell lines cultured in media supplemented with HI‐after EV‐depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS‐derived S‐EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no‐HI FBS and HI‐before EV‐depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell‐derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co‐isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion.https://doi.org/10.1002/jev2.12408extracellular vesiclesfoetal bovine serum (FBS)EV depletion by ultracentrifugationFBS heat‐inactivation |
| spellingShingle | Ornella Urzì Markus Bergqvist Cecilia Lässer Marta Moschetti Junko Johansson Daniele D´Arrigo Roger Olofsson Bagge Rossella Crescitelli Heat inactivation of foetal bovine serum performed after EV‐depletion influences the proteome of cell‐derived extracellular vesicles Journal of Extracellular Vesicles extracellular vesicles foetal bovine serum (FBS) EV depletion by ultracentrifugation FBS heat‐inactivation |
| title | Heat inactivation of foetal bovine serum performed after EV‐depletion influences the proteome of cell‐derived extracellular vesicles |
| title_full | Heat inactivation of foetal bovine serum performed after EV‐depletion influences the proteome of cell‐derived extracellular vesicles |
| title_fullStr | Heat inactivation of foetal bovine serum performed after EV‐depletion influences the proteome of cell‐derived extracellular vesicles |
| title_full_unstemmed | Heat inactivation of foetal bovine serum performed after EV‐depletion influences the proteome of cell‐derived extracellular vesicles |
| title_short | Heat inactivation of foetal bovine serum performed after EV‐depletion influences the proteome of cell‐derived extracellular vesicles |
| title_sort | heat inactivation of foetal bovine serum performed after ev depletion influences the proteome of cell derived extracellular vesicles |
| topic | extracellular vesicles foetal bovine serum (FBS) EV depletion by ultracentrifugation FBS heat‐inactivation |
| url | https://doi.org/10.1002/jev2.12408 |
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